Armelle Martelet

Mass spectrometry (MS) is a tool of choice for the in-depth characterization of new biotherapeutic molecules such as a complex naturally derived trispecific antibody (tsAb) that presents a tyrosine sulfation within the variable domain. Although tyrosine sulfation is an important post-translational modification responsible for strengthening protein-protein interactions, its localization is challenging, as the sulfate group is very labile using conventional positive ion mode fragmentation techniques. In this work, we describe the combination of functional testing and MS-based methods to study the impact of tyrosine sulfation in the tsAb. The presence of sulfation was confirmed by intact mass and peptide mapping analyses. For unambiguous localization of the sulfate group, electron activated dissociation (EAD) MS/MS experiments were employed to generate diagnostic fragments carrying an intact sulfate group. We also demonstrated that a significant decrease in binding of the tsAb to the target antigen was observed following the sulfatase treatment. Taken together, the results from this study support the notion that tyrosine sulfation plays an important role in protein-protein interactions.

Multispecific antibodies, which target multiple antigens at once, are emerging as promising therapeutic entities to offer more effective treatment than conventional monoclonal antibodies (mAbs). However, these highly complex mAb formats pose significant analytical challenges. We report here on the characterization of a trispecific antibody (tsAb), which presents two isomeric forms clearly separated and identified with size exclusion chromatography coupled to native mass spectrometry (SEC-nMS). Previous studies showed that these isomers might originate from a proline cis/trans isomerization in one Fab subunit of the tsAb. We combined several innovative ion mobility (IM)-based approaches to confirm the isomeric nature of the two species and to gain new insights into the conformational landscape of both isomers. Preliminary SEC-nIM-MS measurements performed on a low IM resolution instrument provided the first hints of the coexistence of different conformers, while complementary collision-induced unfolding (CIU) experiments evidenced distinct gas-phase unfolding behaviors upon activation for the two isomers. As subtle conformational differences remained poorly resolved on our early generation IM platform, we performed high-resolution cyclic IM (cIM-MS) to unambiguously conclude on the coexistence of two conformers. The cis/trans equilibrium was further tackled by exploiting the IMⁿ slicing capabilities of the cIM-MS instrument. Altogether, our results clearly illustrate the benefits of combining state-of-the-art nMS and IM-MS approaches to address challenging issues encountered in biopharma. As engineered antibody constructs become increasingly sophisticated, CIU and cIM-MS methodologies undoubtedly have the potential to integrate the drug development analytical toolbox to achieve in-depth conformational characterization of these products.